CN1320974C

CN1320974C - Method for preparing water-soluble nano particles

Info

Publication number: CN1320974C
Application number: CNB031184839A
Authority: CN
Inventors: 何治柯; 梁建功; 谢海燕; 庞代文
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2003-01-21
Filing date: 2003-01-21
Publication date: 2007-06-13
Anticipated expiration: 2023-01-21
Also published as: CN1431070A

Abstract

The present invention discloses a method for preparing water-soluble nano particles. In the method, hydrophilic macromolecules(such as protein, antibodies, antigens, starch, cyclodextrin, avidin, streptavidin, polyethyleneglycol, polyvinyl alcohol, calixarene, etc.) are directly mixed with nano particles with specific fluorescence properties and magnetism in a nonpolar or weak-polar organic solvent; a coating agent is absorbed on coated nano particles by a mechanical grinding method, and the coated nano particles interact with the coating agent further when dissloved in water or a buffer solution after an organic reagent is completely volatilized; pure water-soluble nano particles can be prepared by centrifugally separating the water or the buffer solution with the nano particles twice. The method has the advantages of rapidness, simpleness, convenience, low cost and strong operability, and modified particles can be used as probes to be applied to the fields of chemistry, physics, biology, medicine, etc.

Description

The preparation method of water soluble nanometer particles

Affiliated technical field

The present invention relates to a kind of method for preparing water soluble nanometer particles, affiliated technical field is the interdisciplinary field of subjects such as material, biology, physics, chemistry.

Background technology

In recent years, nano particle has obtained application more and more widely as probe in fields such as biology, physics, chemistry, medical science.These systems overwhelming majority is in the aqueous solution.Yet nano-probe general size distribution directly synthetic in the aqueous solution is all very wide, and some character of probe (as: fluorescence, magnetic etc.) is also undesirable.In order to address these problems, people have been developed the method for organic synthesis.People such as Peng [document Peng Z A, PengX G J Am Chem Soc 2001,123:183～184] are with CdO, Cd (Ac) ₂Salt of weak acid Deng cadmium is a raw material, and adopting with materials such as TOPO is solvent, has synthesized size homogeneous, CdSe semiconductor nano quantum dot that quantum yield is high; We improve its method on this basis, have synthesized the hud typed semiconductor nano quantum dot of CdSe/ZnS of function admirable.People such as Sun [document Sun S H.Zeng H.J.Am.Chem Soc.2002,124:8204～8205] report recently are precursor with the ferric acetyl acetonade, have synthesized the magnetic Nano Fe of size homogeneous in organic facies ₃O ₄Because the product that adopts these class methods to obtain can make the particle diameter of nano particle change in 3～20nm scope, and well solve the agglomeration traits of nano particle after their the change condition.Nano particle outside synthetic in these organic facies is coated with a large amount of hydrophobicity organic molecules, can not directly make probe with the biological substance effect, so will first its surface be modified, is prepared into water miscible nano particle.More existing bibliographical informations nano particle transferred to the method for water from organic facies, as people such as Alivisatos [document Bruchez M Jr.; Moronne M.; Gin P.; Weiss S.; Alivisatos A P.Science.1998,281,2013～2015] reported that silylating reagent coats the semiconductor nano quantum dot after, these semiconductor nano quantum dots can be dispersed in the aqueous solution, and stability is fine, the shortcoming of this method is: operate very complicatedly, required time is long, the cost height; People such as Chan [document Chan W C W.; Nie S M.Science.1998,281,2016～2018] after the foolproof method of employing coats the semiconductor nano quantum dot with TGA, made water miscible semiconductor nano quantum dot, yet, the water-soluble nano quantum dot poor stability that this method prepares is reunited easily.

Summary of the invention

The objective of the invention is, propose the water miscible nano particle method of a kind of new preparation, the water soluble nanometer particles of preparing further is applied to fields such as chemistry, physics, biology, medical science as probe, these field correlation techniques will be made substantial progress.

Nano particle involved in the present invention, refer to the fine particle of particle diameter in 1～100 nanometer, it can be semiconductor nano quantum dot with special photoluminescent property, magnetic nano-particle, metal nanoparticle etc. further are meant the highly sensitive nano-probe in fields such as being applied to chemistry, physics, biology, medical science.The solvent that nanoparticulate dispersed is used can be some nonpolar organic solvents such as n-hexane, benzene, also can be the organic solvent of some low poles such as toluene, chloroform, methyl-sulfoxide, pyridine.

Concrete technical scheme is as follows:

A kind of method (1) for preparing water soluble nanometer particles is scattered in nano particle in the organic solvent, in the mass ratio of nano particle and covering is that 1: 5～100 ratio adds covering, mechanical lapping makes it abundant mixing, and covering is adsorbed on the nano particle.(2) treat (can be blown into Ar gas and quicken its volatilization) after the solvent evaporates, add entry or cushioning liquid, dissolving.(3) be not less than under 18000 rev/mins of high speeds centrifugal, nano particle that big molecule is coated and unnecessary big molecular separation.(4) washing precipitation is dissolved in water or the cushioning liquid for several times again.(5) centrifugal under 3000-5000 rev/min of speed, water soluble nanometer particles is separated with the nano particle of reunion.

According to technical scheme of the present invention, described covering is the big molecules of some hydrophilies, and these hydrophilic big molecules can be protein, antibody, antigen, starch, cyclodextrin, Avidin, streptavidin, polyethylene glycol, polyvinyl alcohol or calixarenes etc.

With the water soluble nanometer particles that protein coats, some has recognition reaction (as: enzyme, antigen, antibody etc.) to chemistry or biological substance, and can keep certain functional activity; Surface active groups is as-NH ₂,-SH ,-COOH etc., can also be used for large biological molecules such as coupled cell, protein, nucleic acid and the little molecule of some medicines, organic molecule, inorganic molecule etc.

The method applied in the present invention is simple, cost is low, and is workable, instrument and equipment that need not be special, and prepared water soluble nanometer particles is widely used, and vast potential for future development is arranged.The water soluble nanometer particles of preparing further is applied to fields such as chemistry, physics, biology, medical science as probe, these field correlation techniques (as: technology such as coding research, high throughput analysis, separation purification) will be made substantial progress.

Description of drawings

Fig. 1 is the TEM image of CdSe/ZnS quantum dot

Fig. 2 coats the emission spectrum of front and back quantum dot for BSA

Fig. 3 is a BSA coated quantum dots fluorescence microscopy image

Fig. 4 is the AFM image (left side is BSA, right quantum dot for the BSA coating) of BSA and BSA coated quantum dots

Fig. 5 for the dextran gel filtration separating resulting of BSA coated quantum dots and BSA (line-dot be the BSA coated quantum dots go out the peak situation, line-square be BSA go out the peak situation)

Fig. 6 be different physiology ion pair BSA coated quantum dots fluorescence intensity influence 1 for control, 2 is Fe ³⁺, 3 is Ca ²⁺, 4 is K ⁺, 5 is Na ⁺, 6 is Mg ²⁺, 7 is Mn ²⁺, 8 is Zn ²⁺, 9 is Cu ²⁺, Cu ²⁺Concentration be 1 * 10 ^-5M, the concentration of other ion is 5 * 10 ^-4(annotate: ferric ion disturbs can be by adding F for M ₊ ^-Eliminate)

Fig. 7 (is followed successively by control, 1 * 10 for the copper ion of variable concentrations from top to bottom to the influence of the fluorescence intensity of BSA coated quantum dots ^-7M, 2 * 10 ^-7M, 4 * 10 ^-7M, 8 * 10 ^-7M, 1.6 * 10 ^-6The Cu of M ²⁺)

Fig. 8 is that the variable concentrations copper ion+(unit is 10 to BSA coated quantum dots fluorescence influence ^-8M)

Fig. 9 is 10 ^-5The Cu of M ²⁺Kinetic curve to the cancellation of BSA coated quantum dots

Figure 10 is prepared water-soluble nano Fe ₃O ₄Uv-visible absorption spectra.

The specific embodiment

Below in conjunction with specific embodiment technical scheme of the present invention is further described

Embodiment 1: water-soluble semi conductor nano-quantum point is as the copper ion selective probe

(1) ZnS coats the preparation of CdSe core-shell type quantum point

With CdO and Se powder is raw material, adopts the method in the document to prepare the CdSe quantum dot.ZnS coats the new method synthetic (this method patent applied for) that the CdSe core-shell type quantum point adopts us to explore by oneself.Transmission electron microscope (TEM) image shows that synthetic core-shell type quantum point particle diameter is evenly distributed, and diameter is about 5nm.The quantum dot quantum yield that different batches synthesizes is between 60%-85%.

(2) bovine serum albumin(BSA) (BSA) coats the preparation of water-soluble quantum dot

The ZnS that will be scattered in the n-hexane coats CdSe core-shell type quantum point methanol extraction; and with methanol wash 2-3 time; remove the unnecessary organic molecule in surface; after being dried under the Ar protection; get in the hexane solution that about 1mg is scattered in 0.5mL again; then about 50mg bovine serum albumin(BSA) (BSA) is put into mortar with the hexane solution of quantum dot; appropriateness is ground and is made it to mix; treat to add after n-hexane volatilizees fully PBS (pH7.4) cushioning liquid of 4mL; make it dissolving; after centrifugal under 18000 rev/mins of speed, abandoning supernatant is removed unnecessary BSA with solution; use the washing precipitation of a small amount of PBS cushioning liquid again; again be dissolved in PBS cushioning liquid then, centrifugal under 5000 rev/mins of speed, remove the quantum dot of reunion.The upper strata is the water miscible CdSe/ZnS solution that BSA coats.

(3) sign of coated quantum dots

Adopt AFM (AFM), fluorescence microscope, fluorescence spectrum etc. that the water-soluble quantum dot of preparation is characterized, inquire into coating mechanism in conjunction with the dextran gel filtration method.The result shows, prepared water-soluble quantum dot good stability, and fluorescence intensity decline is no more than 30% in three weeks.From AFM image, fluorescence microscopy image, fluorescence emission spectrum characterization result as can be known, the quantum point grain diameter after the coating distributes still very even.The several characterization results of analysis-by-synthesis, we think that a quantum dot is coated by 2-4 BSA molecule.

(4) water-soluble quantum dot is as the copper ion selective probe

The alternative copper ion that detects of prepared water-soluble quantum dot.The detectability of copper ion reaches 1 * 10 ^-8M is than low order of magnitude of detectability of the similar detection method of bibliographical information.

Embodiment 2: water-soluble nano Fe ₃O ₄Preparation

Be scattered in the nanometer Fe in the n-hexane ₃O ₄The method of reference literature is synthesized [Sun S H.ZengH.J.Am.Chem Soc.2002,124:8204～8205].To be scattered in the nanometer Fe in the n-hexane ₃O ₄Use precipitation with alcohol, and, remove the unnecessary organic molecule in surface, get about 2mg nanometer Fe with ethanol washing 2-3 time ₃O ₄Again be scattered in the hexane solution of 0.5mL, then will about 50mg bovine serum albumin(BSA) (BSA) with hexane solution put into mortar, appropriateness is ground and is made it to mix, treat to add after n-hexane volatilizees fully PBS (pH7.4) cushioning liquid of 4mL, make it dissolving, with centrifugal under 18000 rev/mins of speed of solution, abandoning supernatant, remove unnecessary BSA, use the washing precipitation of a small amount of PBS cushioning liquid again, again be dissolved in PBS cushioning liquid then, centrifugal under 5000 rev/mins of speed, remove agglomeration nano Fe ₃O ₄The upper strata is the water miscible nanometer Fe that BSA coats ₃O ₄Solution.Figure 10 is prepared water-soluble nano Fe ₃O ₄Uv-visible absorption spectra.

Embodiment 3

To be scattered in the CdSe/ZnS core-shell type quantum dot methanol extraction in the toluene; and with methanol wash 2-3 time; remove the unnecessary organic molecule in surface; after being dried under the Ar protection; get in the toluene solution that about 1mg is scattered in 0.5mL again; then 5mg immunoglobulin (Ig) (IgG) is put into mortar with the toluene solution of quantum dot; appropriateness is ground and is made it to mix; treat to add after toluene volatilizees fully PBS (pH7.4) cushioning liquid of 2mL; make it dissolving; solution is centrifugal with 18000 rev/mins, and abandoning supernatant is removed unnecessary immunoglobulin (Ig); use the washing precipitation of a small amount of PBS cushioning liquid again; again be dissolved in PBS cushioning liquid then, use centrifugally, remove the quantum dot of reunion than slow speed.Supernatant liquor is the water miscible CdSe/ZnS solution that immunoglobulin (Ig) (IgG) coats.

Embodiment 4

To be scattered in the CdSe quantum dot methanol extraction in the chloroform; and with methanol wash 2-3 time; remove the unnecessary organic molecule in surface; after being dried under the Ar protection; get in the chloroformic solution that about 1mg is scattered in 0.5mL again; then about 100mg starch is put into mortar with the chloroformic solution of quantum dot; appropriateness is ground and is made it to mix; treat to add after chloroform volatilizees fully the aqueous solution of 100mL; Ar protection heating down makes it dissolving; solution is centrifugal with 3000 rev/mins of speed, and supernatant is the water miscible CdSe solution that starch coats, and has unnecessary starch not remove in this solution.

Claims

1, a kind of method for preparing water soluble nanometer particles, it is characterized in that this method comprises the steps: that (1) is scattered in nano particle in n-hexane, benzene, chloroform, toluene, methyl-sulfoxide or the pyridine, mass ratio in nano particle and covering is: 1: the ratio of 5-100 adds covering, mechanical lapping makes it abundant mixing, and covering is adsorbed on the nano particle; (2) treat solvent evaporates after, add entry or phosphate buffer solution, the dissolving; (3) be not less than under 18000 rev/mins of high speeds centrifugally, the nano particle that covering is coated separates with unnecessary covering; (4) after the washing of precipitate, be dissolved in again in water or the phosphate buffer solution liquid; (5) centrifugal under 3000-5000 rev/min of speed, water soluble nanometer particles is separated with the nano particle of reunion, supernatant is water soluble nanometer particles;

Described covering is the big molecule of hydrophily, and these hydrophilic big molecules are a kind of in protein, antibody, antigen, starch, cyclodextrin, Avidin, Streptavidin, polyethylene glycol, polyvinyl alcohol or the calixarenes.